This invention relates to a process for producing a transgenic animal wherein gene expression regulatory region for a gene from chromosomal DNA is introduced in the animal to enable the expression of the desired exogenous gene in the animal. More specifically, this invention relates to a process in the field of transgenic animal technology wherein expression of a desired substance in a mammal is enabled by producing a recombinant expression vector capable of amplifying in a host cell such as E. coli by using a DNA fragment including a part of the structural gene coding for mC26 protein and sequences in the upstream and downstream of the mC26 gene that regulate the expression of the mC26 gene; and introducing the gene expression regulatory region for the mC26 gene in the animal by using the thus produced expression vector to produce a model animal for the gene expression regulatory experiment, and to thereby enable the expression of the desired substance in the mammary gland or other tissue of the animal.
In the production of a substance involving use of a genetically manipulated recombinant gene, cultures of microrganisms such as Eschrichia coli, Bacillus subtilis and yeast and various other cells of animal and vegetable origins are used for the host system. The thus produced gene may be introduced in the animal to produce a transgenic animal, and the recombinant gene may be expressed in the transgenic animal to thereby produce the recombinant protein (Palmiter et al., Cell, 29, 701-710, 1982).
In a typical process, a solution containing the recombinant DNA is introduced in the embryo (oosperm) to produce a transgenic animal where the gene introduced is expressed for protein synthesis (DiTullio et al., Bio/Technology, 10, 74-77, 1992). Another process that has been developed involves use of a retrovirus as an intermediate vector (Jaenisch et al., Cell, 24, 519, 1981). This process, however, is associated with a fair risk of oncogene activation and undesirable transcription. Also developed is a process in which the recombinant DNA is directly injected into the animal for the gene expression. This process, when carried out in an industrial scale, is not fully reliable due to insufficiency in gene expression efficiency and stability of the introduced gene.
The recombinant gene introduced in the production of a transgenic animal may be a genomic DNA or a cDNA complimentary to mRNA, and the recombinant DNA sequence introduced may optionally include desired gene expression regulatory regions such as promoter, enhancer, and transcription termination signal regions in the upstream or downstream of the structural gene.
Promoters and enhancer are each provided with different properties, and some lead to expression in a particular organ or tissue (tissue-specific expression) while others lead to expression at a particular timing of the growth (timing-specific expression) or expression in response to the stimulation by environment inside or outside the cell (stimulation-responsive expression). Expression at a desired timing at a desired site in the transgenic animal may be enabled by incorporating gene expression regulatory sequences for the structural gene which are different from the native promoter and the native enhancer upon the preparation of the recombinant sequence to be introduced in the animal (Japanese Patent Application Laid-Open No. 3(1991)-210187).
Various hormone-responsive elements, namely, regions in the gene that respectively respond to stimulus of a hormone such as prolactin, insulin, glucocorticoid, progesterone, estrogen, or the like are associated with alpha casein gene, which is a typical natural milk protein gene. As a consequence, alpha-casein gene is regulated to express in a timing- and tissue-specific manner, namely, to express in the mammary gland during its lactation stage. A recombinant sequence including the gene expression regulatory regions in addition to the milk protein gene undergoes a transcription and a translation as in the case of the natural milk protein gene, and as a consequence, the recombinant sequence is capable of secreting the protein coded by the recombinant gene (Japanese Patent Application Laid-Open No. 63(1988)-291). Secretion into milk of an exogenous gene product by utilizing an expression vector including a gene coding a whey protein (WAP, beta-lactoglobulin) has also been disclosed (Japanese Patent Application Laid-Open No. 63(1988)-291; PCT Application (Japan) Laid-Open No. 64(1989)-500162).
mC26 gene has been identified by Satow et al. (J. Biochem. 99, 1639-1643, 1986) as a gene whose gene product is expressed in mammary gland in a large amount in a lactation-specific and tissue-specific manner. The gene product of mC26 is the product identified by Dowbenko et al. (J. Bio. Chem., 268, 4525-4529, 1993) as leukocyte CAM (cell adhesion molecule), and is referred to as GLYCAM-1. The locus of mC26 has been mapped in mouse, and the nucleotide sequence of the region of the structural gene and a part of the region in the vicinity of the structural gene are already determined (Dowbenko et al., supra.) To an L cell having glucocorticoid receptors derived from mouse fibroblast cell was introduced mC26 gene after its cloning in order to investigate transitional expression and expression in the transformant cell. It was then found that actions of hormones to the mC26 gene is different from those found in conventional cases, and that the transcription product of the mC26 gene is an abnormal RNA that is different from the known mRNA. Difference in the gene expression regulation mechanism of the mC26 gene from those of the known casein genes was thus revealed (Kawamura et al., J. Biochem. 101, 103-110, 1987).